Molecular and Viral Diagnostics Submission Guidelines

Successful isolation and/or detection of viruses in clinical materials depends largely on proper collection and handling of specimens. Care should be taken to protect the virus in specimens from environmental damage and maintain virus infectivity by using the proper transport system.

In general, specimens intended for virological testing should be collected as early as possible in the course of the disease, i.e., within the first 7 days after the onset of illness. Samples collected during the acute phase of viral infection usually contain adequate amounts of virus for detection in available assays. Samples collected later in the course of infection usually require more laboratory time and often yield poor or negative results. Since certain viral infections may predispose the host to secondary viral or bacterial infections, samples collected late in the disease process may lead to a misdiagnosis when secondary infection is involved.

The appropriate samples for virus detection include bodily fluids and secretions (e.g., nasal or conjunctival secretion, genital swabs, urine, saliva, vesicle fluid, semen, milk), feces, blood samples, and skin biopsies from infected, live animals (i.e., antemortem), and relevant tissues and organs from necropsied animals (i.e., postmortem). The collection of antemortem samples from sick animals should be based on clinical manifestation. For example, nasal or nasopharyngeal swabs should be collected from animals with respiratory diseases; fecal samples from animals with enteric diseases; cerebrospinal fluid (CSF), nasal secretion and feces from animals with CNS signs; vesicle fluid and biopsies from animals with skin lesions. The same principle can be applied to the collection of postmortem samples. As a general practice, whole blood* and serum should always be collected from animals with suspected viral diseases, regardless of clinical manifestations. Secondary lymphoid tissues (e.g., tonsil, lymph nodes, spleen) are always good specimens for viral diagnosis.

*For collecting whole blood, citrate is preferable to EDTA as an anticoagulant because EDTA may inactivate some viruses due to its chelating activity.

For best results in isolation and detection of viruses, clinical specimens should be aseptically collected, kept fresh, and transported immediately to the laboratory. If delays are unavoidable or any detrimental affects on virus in samples are anticipated during transport, samples should be refrigerated at 40ºF (4ºC) for no more than 2 days. For longer storage periods, freeze samples at - 70ºC, but NEVER at - 20ºC. Self-defrosting freezers in conventional refrigerators are not appropriate for storage. NEVER freeze whole blood samples. Ideally, frozen samples should be submitted on dry ice, but commercial refrigerant packs can be used if necessary.

Unbleached swabs (e.g., Dacron swabs are available from Baxter) are strongly recommended for collecting nasal and fecal swabs. Standard cotton swabs contain residual bleach that can inactivate viruses. Swabs MUST be prevented from drying. For that reason, swabs may need to be placed in a viral transport system. Ideally, swabs should be placed in a broth medium or balanced salt solution supplemented with 0.5% gelatin, serum, or bovine serum albumin, to protect the viability of viruses in transit plus antibiotics to prevent bacterial and fungal growth. Minimally, physiological saline or Lingo solution could be used on an emergency basis. Specimen collection and transport systems (e.g., Viral Culturette® Becton Dickinson) are available.

It is important to choose not only the most appropriate specimen, but also to collect an adequate amount of specimen for virological testing. Submit a minimum of 3-4 ml serum and 3-5 grams or 5 ml wet volumne of fecal material. Insufficient amounts of sample are a potential cause of inconclusive diagnosis or false-negative result.

When it is necessary to ship a specimen, use a leak-proof container (e.g., tubes, plastic bags) enclosed in a second watertight container containing absorbent material. Ideally, the specimen container should be placed in a Styrofoam box with commercial refrigerant packs or dry ice. Avoid using wet ice because it will melt and leak from the package. Dry ice is preferable if transport requires more than 3 days.

All specimens should be packed in a manner to avoid leakage or breakage, and to withstand the trauma of mailing. Shipping agencies may choose not to deliver leaking packages because of new Department of Transport regulations on shipment of potentially biohazardous materials. In addition, a special permit may be required for interstate transportation of certain veterinary viruses within the United States. Practitioners are referred to published federal guidelines and regulations for details pertaining to packaging, labeling, and interstate shipping of infectious agents (Title 42 CFR Part 72; Title 49 CRF Part 173.386-388).

All specimens should be properly labeled and accompanied by the proper VDL submission form. Please provide all information requested on the form: animal identification, age or body weight, gender, date of onset of disease, major clinical signs, days of gestation if samples originate from abortions, herd size, number of animals affected or dead, date of collection, animal source and location, vaccination history, and differential diagnosis. This latter information is essential for the selection of the most sensitive test system in the laboratory when samples are submitted by mail.

Molecular and Virological Assays

In general, samples appropriate for conventional microbiological evaluation (i.e., detection of viruses) are also suitable for molecular diagnostic tests. This includes excretions and secretions, feces, blood, serum, and biopsies from infected, live animals (i.e., ante mortem), and relevant tissues/organs and washings (e.g., bronchial lavage) from necropsied animals (i.e., post mortem). Refer to the Molecular and Viral Diagnostics Submission Guidelines for comments on sample handling and shipment.

For best results, clinical specimens should be collected aseptically from individual animals. Care should be taken to avoid cross contamination. Samples from different animals can be pooled for molecular assays if cost is a concern, but it is recommended that clients consult with VDL staff prior to pooling samples. Alternatively, samples can be pooled in the lab.

Several molecular diagnostic methods are available for detection or differentiation of viruses, including polymerase chain reaction (PCR), in situ hybridization (ISH), fingerprinting, and sequencing. Some, but not all, molecular techniques first require isolation of the target agent.

Antigen-capturing ELISA (AgELISA)

AgELISA is a rapid, sensitive laboratory diagnostic test for detecting virus or viral antigen(s) in a variety of clinical materials, e.g., tissues, serum, secretions, and excretions. The assay is a variant of the ELISA format in which virus-specific antibody is used to coat the surface of the plates instead of antigen. Therefore, virus or antigen in clinical specimen is captured by antibody. The use of virus-specific monoclonal antibody in AgELISAs provides high specificity. The presence of antibody-antigen complexes is subsequently visualized by a colorimetric reaction. In general, color development is proportional to the amount of viral antigen present in the specimen. One advantage of the AgELISA format is that it detects both infectious and inactivated viruses; however, expense is a consideration. At present, AgELISA is available at the VDL for the rapid detection of:

Fluorescent antibody examination of frozen tissue section (FATS)

FATS is a rapid, specific laboratory diagnostic test for detecting viral antigen in tissues and cell smears. The test utilizes virus-specific antibody labeled with a fluorochrome. When viewed with a fluorescence microscope, complexes of viral antigen and labeled antibody appear as fluorescent green areas. For best results, FATS requires very fresh clinical specimens. Freezing and thawing tissues can be detrimental to the test. If autolysis of tissues is anticipated, other antigen detection methods, such as immunohistochemistry (IHC) on formalin-fixed tissues, should be considered.

It is important to recognize that VI and EM are nonspecific tests that can detect a variety of viruses. In contrast, FATS and IHC are very specific tests that, because they are use virus-specific reagents (e.g., antibody), only detect the targeted virus. Furthermore, FATS and IHC cannot be performed on certain specimens, such as serum or feces.

Polymerase chain reaction (PCR)

PCR is a process by which a portion of viral nucleic acid (DNA or RNA) is replicated a million times or more. Detection of this amplified product (amplicon) indicates that the sample is positive for the target virus.

Proper amplification relies on a set of two short synthetic oligonucleotides (primers). Good test performance requires that primers bind only to the corresponding nucleotide sequences of the viral genome and nothing else. Thus, the specificity of the assay and accuracy of the results depend upon the design of PCR primers. Theoretically, a PCR-based assay is capable of detecting one copy of the viral genome. However, depending upon the target agent, type of sample, and condition of sample, diagnostic sensitivity often is not as sensitive as anticipated and, depending on the circumstances, conventional assays may provide equivalent test performance. Also, PCR is expensive relative to most other diagnostic tests and positive results do not always have biological significance, i.e., PCR reacts with inactivated viruses, as well as infectious viruses.

There are several types of PCR assays:

RT-PCR is used to detect target RNA from clinical specimens. Reverse transcription (RT) of RNA is required to make complementary DNA for further amplification. This assay is most frequently used for specific detection of RNA viruses.

Nested PCR is a PCR done in two steps, a primary PCR reaction and a nested reaction. The primary (or first) reaction uses a set of primers to generate a product that serves as the template for the nested (or second) reaction. The nested reaction uses a set of PCR primers specific for a region within the amplified product from the first reaction. Therefore, the nested reaction often serves as a confirmation for the specificity of the PCR products amplified in the primary reaction.

Multiplex PCR is a PCR designed to detect more than one target sequence in a single PCR reaction. The assay uses two or more sets of primers. Each set of primers is specific for a different target sequence. The assay is most commonly used for simultaneous detection of multiple viral genes and differentiation of genotypes or subtypes of related microorganisms.

Real-time PCR combines PCR amplification and detection into a single step. The basic principle of real-time quantitative PCR is the detection of target sequences using a fluorogenic 5' nuclease assay (often called "TaqMan"). The advantages of this system include high reproducibility, the capability of handling large numbers of samples, the potential for quantitative results, and decreased turnaround time. The disadvantages include high instrument cost and the requirement for technical proficiency.

Virus isolation

Virus isolation consists of two steps - the attempted recovery of virus and identification of the isolate. Isolation is attempted using in vitro cell culture. Identification of the isolate is done using immunofluorescence microscopy, electron microscopy, or molecular techniques. Although isolation of virus followed by identification is considered to be the definitive diagnosis, VI is often laborious, expensive, and time consuming. Results are not generally available for 1-2 weeks after submission. Therefore, VI should primarily be attempted under specific circumstances:

When other detection methods fail or when trying to isolate virus(es) from previously unrecognized diseases.

If there is no other detection method of similar or greater sensitivity.

If the virus is required for other purposes, such as differentiation (e.g., RFLP, sequencing), characterization (e.g., typing), and/or for production of autogenous vaccines.

Aseptic collection and proper handling of clinical specimens is critical for VI. Although certain viruses (e.g., parvovirus, circovirus) can withstand harsh environmental changes, this is not true for most viruses, particularly enveloped viruses. Virus isolation can be done on most clinical specimens, including biopsy and necropsy tissues, blood, secretions, and excretions. However, some clinical materials, e.g., urine, feces and semen, are difficult to work with because they are toxic to cell cultures.

The quantity of sample required for VI is usually more than that needed for rapid diagnostic assays so collect and submit the adequate amount of sample. To avoid unnecessary expense, be specific in your VI requests.

Molecular Assays for Differentiation and Genetic Characterization

Differential PCR can sometimes be used to distinguish closely related targets. Differential PCR is done either in a multiplex format using two or more sets of primers or by running two separate PCR assays. Refer to the ISU VDL Fee Schedule for a list of differential PCRs.

RFLP analysis is a molecular differential technique that, to a limited extent, can distinguish between two viruses at the genomic level. RFLP is based on the fact that restriction enzymes recognize specific nucleotide sequences and cut the genome at that location. In general, the RFLP procedure consists of isolating the target microorganism from a clinical specimen, extracting DNA or RNA, digesting the nucleic acid material with restriction enzymes, and gel electrophoresis of the resulting products. The pattern of fragments observed on the gel is used to characterize or compare isolates. In general, RFLP requires virus isolates; however, RFLP using PCR products instead of native nucleic acid has recently been developed. This method provides faster turnaround since it is not necessary to isolate and propagate virus.

RFLP is useful for differentiating minor differences at the strain level that normally cannot be detected by any antigenic assays, such as tissue immunoassays, serology, and enzyme immunoassays. RFLP analysis is rapid and less expensive than sequence analysis, but RFLP will miss many of the genetic differences that are revealed by sequencing.

Sequence analysis is a molecular tool for use in characterizing the genetic information of microorganism in detail. Sequencing provides a list of the nucleotides in the viral genome in the order in which they appear. Comparison of the genetic information makes complete differentiation between viruses possible. Either partial or whole genomic sequencing can be done, depending upon the size of genome and purpose of the analysis. In addition, RFLP patterns and amino acid composition can be predicted from sequence data. One disadvantage of sequencing is the expense of conducting the analysis.